DNA double-strand break (DSB) repair is a central process in genome maintenance, broadly divided into homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways. Of these, NHEJ, the direct ligation of DSB ends, is most likely to execute the chromosomal rearrangements that cause cancer because such junctions typically lack extensive homology. NHEJ is also a genome caretaker that promotes accurate repair of DSBs, however, underscoring its dichotomous role in genome (in)stability. Prior work has led to the apparent identification of nearly all eukaryotic NHEJ proteins. These include: (i) the structural end- binding protein Ku; (ii) DNA ligase IV, comprised of its catalytic subunit (Lig4/Dnl4) and two supporting proteins (XRCC4/Lif1 and XLF/Nej1); and (iii) end processing polymerases of the Pol X family (Pol <, Pol ;/Pol4). Many features of these various proteins are also known, including substantial structural information. What is not known is how they interact with each other and the DNA to achieve the dynamic process of repair. There is an extensive protein architecture used during NHEJ with currently little insight into how its parts assemble onto the limiting DSB substrate in both space and time. Once there, NHEJ enzymes use poorly understood mechanisms to overcome the unique challenge of catalyzing reactions on a DNA substrate comprised of unstably associated halves. This project will explore these outstanding issues using powerful and novel genetic assays in the budding yeast model organism, with four specific aims addressing: (i) the interactions between Ku and DNA ligase IV that recruit and productively position the ligase for catalysis; (ii) the specific and multiple functions of the DNA ligase IV BRCT domains in supporting NHEJ; (iii) the specific features of Pol X family DNA polymerases that allow only them to catalyze certain synthetic events during NHEJ; and (iv) similar specific features of catalysis by DNA ligase IV that optimize its ability to join DSB ends. PUBLIC HEALTH RELEVANCE: Nonhomologous end joining (NHEJ) of double-strand breaks is a key DNA repair process that maintains the genome but also paradoxically executes chromosomal rearrangements. Because of this dichotomous action, a detailed description of the NHEJ reaction mechanism is required to understand the potential consequences of NHEJ deficiency as might occur during oncogenesis or by inhibition as a potential therapeutic intervention during the treatment of cancer.